Unlike opportunistic pathogens, the fungal pathogen Histoplasma capsulatum is not cleared, nor controlled, by the innate immune system. Current knowledge about the molecular mechanisms that underlie Histoplasma pathogenesis remains limited as only a few virulence factors have been identified and characterized to date. Our preliminary studies indicate that specific factors modified by O-linked mannosylation facilitate Histoplasma virulence in vivo. Although a number of glycosylated fungal proteins have been studied, the role of post-translational modification of such proteins in facilitating fungal pathogenesis is not well understood. To provide a foundation for mechanistic studies of the role of O-linked mannosylation, we will determine at which stage of infection or against which immune defense effectors O-linked glycoproteins act to enhance the survival of Histoplasma yeasts. To identify the specific glycoproteins with pathogenesis roles, we will use bioinformatics and glycoproteomics to first define the O-linked glycoproteins produced by pathogenic-phase Histoplasma cells. Secondly, using genetically engineered strains lacking specific mannosyltransferase enzymes, we will define which proteins serve as substrates for each mannosyltransferase. Finally, as virulence attenuation is linked to loss of certain mannosyltransferases, we will use the substrate assignments to prioritize glycoproteins as new candidate virulence factors that enable Histoplasma to survive innate immune defenses during mammalian infection.